tbuckley/cabot-search huggingface dataset:
Abstract P1138: Combination Of Pluripotency And Cardiac Transcription Factors Reprogram Skin Fibroblasts Into Cardiomyocytes
Authors: Waqas Ahmad, Rida-e-Maria Qazi, Rabbia Muneer, Irfan Khan, Asmat Salim, Mohsin Khan
Journal: Circulation Research (2022)

Direct reprogramming of autologous somatic cells into cardiomyocytes is a novel approach which is being employed for cardiac regeneration. Multiple studies have combined different cardiac-specific factors that could directly reprogram cardiac fibroblasts into induced cardiomyocyte-like cells. However, this approach needs heart biopsy for the isolation of autologous fibroblasts which involves extensive surgical procedures that require precision. In this study, we aimed to develop an approach which can directly reprogram somatic cells into cardiomyocyte-like cells using a combination of pluripotency and cardiac transcription factors. Skin fibroblasts were isolated from rat neonatal pups and were induced into cardiomyocyte-like cells using non-viral integration of cardiac transcription factors (GATA4, Mef2c and Nkx2.5) and OKSIM plasmid carrying the iPSC factors, Sox2, Oct4, Klf4 and cMyc. After 72 h, cells were analyzed for the expression of cardiac markers by qPCR and immunocytochemistry. Gene expression analysis showed significantly higher expression of cardiac markers GATA4, cMHC, Mef2c, cTnT, cTnI, and Nkx2.5 Immunostaining confirmed the expression of cardiac proteins GATA4, cMHC, cTnT, cTnI, and Nkx2.5. These results imply that the transfected cells started differentiating towards cardiac lineage. Transfected cells were also transplanted in rat myocardial infarction (MI) model immediately after ligation of left ventricle descending (LAD) artery. After 2 and 4 weeks of cell transplantation, the animals were assessed for cardiac function via echocardiography. They showed significant improvement in the cardiac function as compared to MI and non-transfected groups. After 4 weeks, the hearts were harvested, and histological analysis was performed for the assessment of fibrosis and left ventricular wall thickness. Rats transplanted with transfected cells showed significantly reduced fibrosis and increased wall thickness as compared to MI and non-transfected groups. In conclusion, transient and combined expression of cardiac transcription and iPSC factors in neonatal somatic cells leads to the transdifferentiation of skin cells into myogenic lineage. This approach can be useful for future therapeutic application for cardiovascular diseases.
Abstract P3055: Combined Metabolomic And Transcriptomic Profiling Approaches Reveal The Cardiac Response To High-fat Diet
Authors: John P Morrow, Leroy C Joseph, Quynh Nguyen, Jianting Shi, Hanrui Zhang
Journal: Circulation Research (2022)

The response of vital organs to different types of nutrition or diet is a fundamental question in physiology. It is unclear how different diets can alter cardiac metabolism and function. We examined the cardiac response to 4 weeks of high-fat diet in adult mice, measuring cardiac metabolites and mRNA. Metabolomics showed dramatic differences after high-fat diet, including increases in several acyl-carnitine species. The RNAseq data showed changes consistent with adaptations to use more fatty acid as substrate and an increase in the anti-oxidant protein catalase. Changes in mRNA were correlated to changes in protein level for several highly responsive genes, including catalase and HMGcs2. We also found significant sex differences in both metabolomics and RNAseq datasets, both at baseline and after high fat diet. This work reveals the response of a vital organ to dietary intervention at both metabolomic and transcriptomic levels, which is a fundamental question in physiology that may have relevance for cardiovascular diseases. This work also reveals significant sex differences in cardiac metabolites and gene expression. Figure 1: Metabolomic changes after HFD; electron microscopy shows changes in mitochondrial morphology. A. Principal component analysis (loading) of metabolites demonstrates that the first two principal components (PC) cluster the samples into 4 groups. PC1 + PC2 account for 85.47% of the proportion of variance in this metabolomic dataset.B. Graph of acyl-carnitine species, dots with lines indicated mean +/- SEM. “C#” refers to the number of carbon atoms in the lipid, that is C16 is palmitoylcarnitine, C14 is myristoylcarnitine, etc. N= 10 hearts for each group. The comparison of control (chow) to HFD is highly statistically significant by 2-way ANOVA, p&lt;0.0001.C. Representative examples of electron microscopy from cardiac ventricular samples.D. Graphs of average mitochondrial area (um 2 ) and average number of mitochondria per high-power image. N= 4 hearts for each group, * indicates significantly different by t-test.
Abstract EC101: Premature Stop Mutations In <i>Mybphl</i> Share The Common Pathomechanism Of Preventing Protein Incorporation Into The Cardiac Myofilament
Authors: Alejandro Alvarez-Arce, Kelly Araujo, Lucas Wittenkeller, David Y Barefield
Journal: Circulation Research (2022)

Cardiomyopathy and arrhythmia are diseases with significant genetic linkage. Cardiac myosin binding protein-C (cMyBP-C) is a myofilament associated protein with a C-terminal composed of repeating immunoglobulin (Ig) and fibronectin III (FnIII) domains that bind to the myosin thick filaments and titin. Truncating mutations in the C-terminal of MyBP-C result in improper myofilament incorporation and degradation of the truncated protein and is a major cause of hypertrophic cardiomyopathy. Myosin-binding protein-H like ( MyBP-HL ) is a protein that was recently identified that is structurally related to MyBP-C. Both cMyBP-C and MyBP-HL contain repeated C-terminal Ig and FnIII domains that bind to myosin. MyBP-HL is different in that it is primarily expressed in the atria where it interacts with the myofilament. MyBP-HL premature stop mutations are associated with improper myofilament incorporation, and has been linked to ventricular conduction system abnormalities, atria enlargement, and dilated cardiomyopathy (DCM) in humans. The gnomAD database reports 9 stop-gain mutations for MYBPHL in humans including: Gln29, Trp54, Arg113, Tyr123, Trp158, Trp192, Lys250, Arg255 and Tyr307. Prior investigation modeled the MyBP-HL Arg255X mutation and showed no MyBP-HL localization into the myofilament. A null mouse model showed that loss of MyBP-HL resulted in arrhythmia and cardiomyopathy, similar to that observed in humans. We hypothesize that all MyBP-HL truncation mutations will share a common mechanism by failing to produce MyBP-HL that integrates into the sarcomere. We test this hypothesis by transfecting neonatal rat cardiomyocytes with MyBP-HL constructs containing these mutations. We tested Trp54, Arg113, Tyr123, Trp158, Trp192, Lys250 and Arg255 MyBP-HL mutations. We measured co-localization events between MyBP-HL mutants and MyBP-C in atrial and ventricular overexpressed cells and observed that stop gained mutations disrupt normal localization as we expected. Moreover, we tested wild-type MyBP-HL overexpression, and both showed to be able to incorporate in a similar pattern to cMyBP-C C-zone doublets. These truncating mutations in MYBP-HL may increase risk for developing arrhythmias, atria enlargement and DCM.
Abstract P2050: Acetyl-coa Synthesis Is Required For Chromatin Remodeling In Myofibroblast Differentiation And Persistence
Authors: Michael P Lazaropoulos, Allison Reiter, Andrew A Gibb, Rajika Roy, John W Elrod
Journal: Circulation Research (2022)

Heart failure (HF) features fibrotic remodeling by myofibroblasts, a differentiated fibroblast population responsible for wound repair. Discovering mechanisms of myofibroblast formation and function may yield novel targets to delay or reverse HF. Myofibroblasts metabolically reprogram to mediate transcriptional activation of fibrotic genes through chromatin remodeling. We previously reported that α-ketoglutarate production for histone demethylation is essential for cardiac fibrosis, but metabolic regulation of histone acetylation in fibrosis is unclear. ATP-citrate lyase (ACLY) and Acetyl-coenzyme A synthetase 2 (ACSS2) synthesize acetyl-CoA in the cytoplasm and have also been reported to supply substrate for histone acetyltransferases in the nucleus to modify regulatory histone residues. To investigate acetyl-CoA metabolism in myofibroblast formation, we stimulated adult mouse and human cardiac fibroblasts (CFs) with the pro-fibrotic agonist transforming growth factor β (TGFβ) and treated cells with pharmacological inhibitors of ACLY and ACSS2. Either ACLY or ACSS2 inhibition sufficiently reverted differentiated myofibroblasts to a non-fibrotic phenotype, even during continuous TGFβ stimulation. Genetic deletion of ACLY in fibroblasts recapitulated these results. Further, ACLY inactivation was sufficient to decrease global histone acetylation. ACLY deletion, specifically in activated myofibroblasts in the adult mouse heart, slowed LV functional decline and remodeling in a model of pressure overload HF. Our data indicate that ACLY and ACSS2 are necessary for myofibroblast differentiation and persistence. We hypothesize that acetyl-CoA synthesis is necessary for histone acetylation required for activation of myofibroblast genes. Ongoing work will determine the molecular mechanisms of both enzymes in chromatin remodeling and in cardiac injury. In summary, acetyl-CoA producing enzymes are attractive clinical targets to reverse cardiac fibrosis.
Abstract P1142: Engineering Vascular Therapies With Angiogenic Factors For Cardiac Regeneration And Tissue Remodeling
Authors: Alicia Minor, Stephanie Roser, Keyana Zahiri, Elena Song, Momoka Kobayashi, Collin Polucha, Kareen L Coulombe
Journal: Circulation Research (2022)

The limited regenerative capacity of the adult human heart presents a critical need to stabilize and support injured heart muscle to mitigate the progression of heart failure. The effectiveness of remuscularization therapies using human induced pluripotent stem cell derived cardiomyocytes depends host mediated angiogenesis to direct the formation of stable, perfused vascular networks within the engineered cardiac implant. We hypothesize that potent combinations of growth factors will stimulate angiogenesis in the myocardium and increase vascular development in engineered cardiac tissue. In a fractional factorial experiment, we assessed the effect of ten growth factors, cytokines and enzymes: VEGF, bFGF, Shh, PDGF, IGF-1, EGF, TGF-β1, Ang-1, MCP-1 and MMP-9 on in vivo angiogenesis in a subcutaneous Matrigel plug assay in the rat. Five factors were highly potent by metrics of vessel density and vascular cell recruitment in explants and warranted further investigation. We then designed a two-level, fractional factorial experiment using a defined collagen-alginate gel to deliver high/low doses of these 5 factors subcutaneously to elucidate main effects and two-way interactions that regulate vessel development in vivo . After 7 days, Griffonia Simplicfolia lectin I was administered to label perfused vasculature then explants were removed and processed for immunohistochemistry. Our data show that 3 unique cocktails yielded a 10-fold increase in lumen density, a 3.3-fold increase in lumen size, and a 3.7-fold increase in endothelial cell recruitment relative to unloaded controls. Ongoing analyses will determine which angiogenic cocktail to deliver in a customizable biomaterial-based vascular therapy for applications in the heart. This novel therapy will be delivered with an engineered cardiac tissue onto the epicardium in a rat I/R MI model to quantify vascular remodeling in the implant and underlying host tissue and whole heart function over 4 weeks. In conclusion, we have identified five potent angiogenic factors for modulating revascularization responses and our ongoing work aims to understand the potential synergy between revascularization and remuscularization therapies for heart regeneration.
Abstract P3043: Functional Role Of Endothelial Mitochondrial Oxidative Stress In Cognitive Impairment: Effects Of L-arginine In Hypertensive Frail Older Adults
Authors: Pasquale Mone, Stanislovas S Jankauskas, Fahimeh Varzideh, Urna Kansakar, Angela Lombardi, Gaetano Santulli
Journal: Circulation Research (2022)

Background: Hypertension is common in older adults, particularly in frailty. The association of hypertension and frailty leads to a high-risk of cognitive impairment in older adults. Endothelial dysfunction has been shown to underlie both hypertension and cognitive impairment. L-Arginine is an amino acid involved in many biological processes and is a substrate of two enzymes: nitric oxide (NO) synthase (NOS) and arginase (NOA). L-Arginine is fundamental for NO production by endothelial cells, regulating vascular tone and cardiovascular homeostasis, improving inflammation and oxidative stress. On these grounds, we hypothesized that L-Arginine could improve cognitive impairment via its effect on endothelial mitochondria. Methods: To assess the effects of L-Arginine on endothelial oxidative stress and cognitive function, we used an in vitro and a clinical approach. First, we treated with L-Arginine human umbilical vein endothelial cells (HUVECs). HUVECs were cultured in EGM-2 medium and incubated at 37°C and 5% CO 2 . HUVECs were plated on glass bottom culture dishes and treated with Angiotensin II (1 μM) and Angiotensin II with L-Arginine (500 μM) in EGM-2 medium for 24h. The generation of reactive oxygen species (ROS) was quantified using MitoSOX™ Red (an established ROS probe that accumulates in mitochondria), incubating cells for 10 min at 37°C and 5% CO 2 , as we previously described. We then designed a single-blind, placebo-controlled, parallel-group, randomized trial to observe the effects of 4-weeks oral supplementation of L-Arginine on global cognitive function of hypertensive frail older patients. Results: In vitro , we demonstrated that L-Arginine significantly attenuated Angiotensin II-induced oxidative stress in endothelial cells. Co-treatment of HUVECs with Ang II and L-Arginine significantly attenuated mitochondrial ROS production (p&lt;0.001). In the clinical study, we evaluated 35 frail hypertensive elderly patients assigned to L-Arginine and 37 assigned to placebo: at follow-up, we found a significantly improved Montreal Cognitive Assessment (MoCA) test score in the L-Arginine treated arm compared to placebo (p&lt;0.05). Conclusions: Taken together, our findings indicate for the first time that oral L-Arginine supplementation improves cognitive impairment in frail hypertensive older adults through a mechanism that involves endothelial mitochondrial ROS.
Abstract P2051: Incomplete Regression And Persistence Of Interstitial Fibrosis Following Removal Of Left Ventricular Pressure Overload
Authors: Lily S Neff, An O Van Laer, Catalin F Baicu, Michael R Zile, Amy Bradshaw
Journal: Circulation Research (2022)

Background: Left ventricular pressure overload (LVPO) can develop from antecedent conditions, like aortic stenosis, and is associated with increased myocardial collagen content that can lead to increases in myocardial stiffness. Alleviation of LVPO in patients undergoing surgical aortic valve replacement (SAVR) resulted in reduced but persistent fibrosis evident in biopsies taken 5-yrs following SAVR. Little is known regarding cellular mechanisms that contribute to ECM turnover following hemodynamic unloading. Objective: Determine the time course of ECM remodeling after alleviation of LVPO. Methods: Transverse aortic constriction (TAC) was used to induce LVPO. Following 4wks of TAC, alleviation of LVPO was performed by removing the TAC band (unTAC). Five time points were assessed: Control no TAC, 4wk TAC, 4wk TAC+2wk unTAC, 4wk TAC+4wk unTAC, and 4wk TAC+6wk unTAC for cardiomyocyte cross-sectional area (CSA), collagen volume fraction (CVF), collagen degradation, and myocardial stiffness. Results: CSA increased by 47% at 4wk TAC vs control. Compared to 4wk TAC, CSA decreased by 20% at 2wk unTAC, but returned to control levels by 6wk unTAC. CVF increased by 204% at 4wk TAC vs control. Although CVF was similar between 4wk TAC and 2wk unTAC, 2wk unTAC demonstrated robust increases in markers of collagen degradation (cathepsin K, pro-matrix metalloproteinase 2 (pro-MMP2), MMP3, and MMP8). CVF significantly decreased at 4wk unTAC compared to 2wk unTAC. However, the markers of collagen degradation were reduced by 4wk- and 6wk unTAC likely contributing to persistent fibrosis. Furthermore, passive stiffness remained elevated at 6wk unTAC. Conclusion: In LVPO hearts, hypertrophy and fibrosis were present. Although cardiomyocyte hypertrophy fully regressed with normalization of hemodynamic load, increased myocardial stiffness and elevated interstitial fibrosis were persistent.
Abstract P2017: Proximity Proteomic Mapping Of Cav1.2 In Systolic Heart Failure
Authors: Jared Kushner, Guoxia Liu, Elaine Y Wan, Steven Marx
Journal: Circulation Research (2022)

Introduction: In heart failure (HF), there is dysregulation of Ca V 1.2. This includes reduced expression, pathologic splice variation, and loss of β-adrenergic activation. We showed that adrenergic-induced inotropy requires PKA phosphorylation of Rad and disinhibition of Ca V 1.2. We hypothesize that proximity labeling Ca V 1.2 can illuminate important pathobiology in HF, such as whether loss of adrenergic activation follows absence of Rad from the channel nanodomain. Methods: Transgenic (TG) mice with cardiac expression of Ca V 1.2 subunits fused to the engineered ascorbate peroxidase, APEX2 were made. HF was confirmed by echocardiography 3 months after coronary artery ligation. Multiplexed quantitative mass spectrometry was performed on homogenates of APEX2-catalyzed proximity labeled myocytes. Results: After surgery, 5 TG Ca V 1.2 α 1C -APEX and 5 β 2B -APEX mice had FS less than 25%. The biotinylated proteins from the HF samples were compared to biotinylated proteins from 6 healthy α 1C -APEX or β 2B -APEX mice. More than 2000 proteins were quantified. We found significant enrichment of microtubule proteins (blue), the 26s proteasome (red), as well as key regulatory proteins such as Rad and PKA (green). Conclusions: We demonstrate that proximity proteomics has the power to probe deeply, in vivo , the molecular biology of HF at nanometer resolution. Attenuated adrenergic responses do not follow absence of PKA or Rad from proximity to Ca V 1.2, and we identified a potential mechanism for loss of channel in HF—increased proteosomal degradation. Future studies will explore whether Rad is properly released from stimulated channels in HF as well as mechanisms of channel degradation.
The effect of oral midazolam and diazepam on respiration in normal subjects
Authors: KH Mak, YT Wang, TH Cheong, SC Poh
Journal: European Respiratory Journal (1993)

Benzodiazepine have been shown to suppress ventilatory responses to hyperoxic hypercapnia (HCVR) and isocapnic HVR when taken parenterally. Most patients would, however, prefer to take an oral rather than parenteral preparation but the effect of oral benzodiazepine on these ventilatory responses has not been well studied. We therefore studied the effect of oral midazolam (7.5 mg) and diazepam (5 mg) both given orally on resting ventilation and respiratory drive, as assessed by HCVR and HVR. Flumazenil, a specific benzodiazepine antagonist, was administered intravenously to reverse the effect. A mental alertness-drowsiness index in five grades, from 1 (awake and alert) to 5 (asleep), was used to assess the sedation effect. Six normal male subjects, (aged 31 +/- 1.6 yrs) (mean +/- SD), participated in the study. Mean resting ventilation, and ventilatory response to HCVR and HVR were not significantly altered by these drugs when taken orally. Flumazenil also had not significant effect on HCVR and HVR. However the mental alertness-drowsiness index rose from 1 to 2.83 with oral midazolam and reversed to 1.25 with flumazenil. Similarly, this index increased from 1 to 2.25 after oral diazepam and reversed to 1.42 after flumazenil. In conclusion, we found that even though oral midazolam and diazepam produced a significant sedation effect, which was reversed with flumazenil, the drugs had no effect on ventilation at rest and the ventilatory responses to hypoxia and hypercapnia.
Abstract P2113: A Novel Patient-derived Stem Cell Model Of Restrictive Cardiomyopathy Exhibits Impaired Cardiomyocyte Relaxation Secondary To Increased Myocardial Calcium Sensitivity
Authors: David Staudt, Ricardo Serrano, Dries Feyen, Mark Mercola
Journal: Circulation Research (2022)

Introduction: Restrictive Cardiomyopathy (RCM) is a rare disease characterized by profound, isolated defects in myocardial relaxation, normal or near-normal myocardial wall thickness, and preserved contractile function. Outcomes for patients with RCM are poor, with increased morbidity and mortality compared to other forms of cardiomyopathy. In some patients, RCM can be caused by genetic mutations, notably in genes encoding for components of the sarcomere. While many mutations have been described in RCM patients, the molecular and cellular mechanisms leading from these mutations to the patient’s physiological phenotype are poorly described. Hypothesis: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) derived from RCM patients will recapitulate the myocardial relaxation defects seen in RCM and provide a novel in vitro model for investigating the molecular mechanisms underpinning this disease. Methods: Induced pluripotent stem cells (iPSCs) were derived from a pediatric patient with RCM caused by a TNNT2 R94C mutation. CRISPR was used to correct the mutation, and cellular mechanics and calcium handling properties were compared to the patient line using a novel high-throughput traction-force imaging platform. Transcriptional profiles were compared using RNA-seq. Results: RCM cells carrying the TNNT2 R94C mutation show increased calcium sensitivity leading to increased resting tension at baseline, as well as prolonged calcium kinetics leading to prolonged relaxation times. Gene expression profiling reveals changes in expression of ECM genes, which may further contribute to the impaired myocardial relaxation seen in RCM. Conclusions: Patient-derived hiPSC-CMs from an RCM patient recapitulate the relaxation defects seen in RCM and highlight the central role of increased calcium sensitivity in impaired myocardial relaxation.